1.Field of the Invention
The present invention concerns improvements in the purification, testing and therapeutic use of heparin-binding proteins, based upon the inhibition of the interaction between such proteins and naturally occurring heparin-like substances. More specifically, the invention relates to improved methods and compositions for the administration of heparin-binding proteins. The invention further relates to improved methods for the purification and for testing the biological activity of heparin-binding proteins.
2. Description of Background and Related Art
It is known that a large variety of naturally occurring, biologically active polypeptides bind heparin. Such heparin-binding polypeptides include cytokines (also termed chemokines), such as platelet factor 4 and IL-8 (Barber et al., Biochim. Biophys. Acta 286, 312-329 (1972); Handin et al., J. Biol. Chem. 251, 4273-422 (1976); Loscalzo et al., Arch. Biochem. Biophys. 240, 446-455 (1985); Zucker et al., Proc. Natl. Acad. Sci. USA 86, 7571-7574 (1989); Talpas et al., Biochim. Biophys. Acta 1078, 208-218 (1991); Webb et al., Proc. Natl. Acad. Sci. USA 90, 7158-7162 (1993)); heparin-binding growth factors (Burgess and Maciag, Annu. Rev. Biochem. 58, 576-606 (1989); Klagsbrun, Prog. Growth Factor Res. 1, 207-235 (1989)) , such as epidermal growth factor (EGF); platelet--derived growth factor (PDGF); basic fibroblast growth factor (bFGF); acidic fibroblast growth factor (aFGF); vascular endothelial growth factor (VEGF); and hepatocyte growth factor (HGF) (for the latter see also Liu et al., Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G642-G649 (1992)); and selectins, such as L-selectin, E-selectin and P-selectin (Norgard-Sumnicht et al., Science 261, 480-483 (1993)) .
The preparation of homogeneously sized, heparin-derived oligosaccharides prepared from heparin has been reported, and heparin-derived hexa- and octasaccharides were described to be able to inhibit the interaction between cell surface heparan proteoglycan and bFGF (Ishihara et al., J. Biol. Chem. 268, 4675-4683 (1993)). The structure of a specific heparin-derived hexasaccharide showing high affinity for bFGF was described by Tyrrell et al., J. Biol. Chem. 268, 4684-4689 (1993).
The heparin-binding proteins are typically cleared rapidly from the plasma in vivo, and this rapid clearance greatly limits their therapeutic applications. Although the liver and the kidney have been identified as the major clearance organs for a number of heparin-binding proteins (see, e.g. Kim et al., J. Pharm. Sci. 77, 200-207 (1988); Sugiyama et al., Pharm. Res. 6, 194-204 (1989), Sugiyama et al., J. Controlled Release 13, 157-174 (1990) and Sugiyama et al., Receptor Mediated Hepatic Clearance of Peptide Hormones, In: Topics in Pharmaceutical Sciences 1989, Breimer et al., eds., Elsevier, New York, 1989, p. 429-443), little is known about the mechanism of their rapid removal from the circulation. In a recent publication (Liu et al., supra) it was proposed that there are at least two different kinds of binding sites for HGF on the surface of hepatic cells: a heparin-washable binding site with lower affinity and a heparin-resistant, acid-washable binding site with higher affinity, and that the low-affinity binding site appears to play a role in the internalization of HGF. The binding of other heparin-binding proteins to their respective cell surface receptors was described to be dependent upon heparin-like molecules (see, e.g. Gitay-Goren et al., J. Biol. Chem. 267, 6093-6098 (1992) for VEGF), and it was shown that the addition of exogenous heparin is able to potentiate the binding of certain heparin-binding proteins to their receptors.
It would be desirable to increase the plasma half-life and/or decrease the plasma clearance of heparin-binding proteins.
It would further be desirable to improve the therapeutic potency of heparin-binding proteins.
It would also be desirable to improve the bioavailability of such polypeptides when they are not directly administered into the blood stream.
It would additionally be desirable to prevent the inactivation of heparin-binding polypeptides by other binding proteins in blood.
It would further be desirable to enhance the sensitivity of assays for the detection of heparin-binding proteins.
It would be also desirable to enhance the purification of heparin-binding proteins from cell cultures.
Accordingly, it is an object of the present invention to provide a method for extending the plasma half-life of heparin-binding proteins.
It is another object of the invention to improve the therapeutic potency of heparin-binding proteins.
It is a further object, to improve the bioavailability of heparin-binding proteins.
It is a still further object to provide methods for preventing the inactivation of heparin-binding proteins by other blood proteins.
It is yet another object to provide new and improved pharmaceutical compositions of heparin-binding proteins.
It is an additional object to enhance the sensitivity of assays for the detection of heparin-binding proteins.
It is a still further object to facilitate the purification of heparin-binding proteins.
It is another object of the present invention to provide methods for the intraperitoneal or subcutaneous administration of heparin-binding proteins.